Jon Hanley Flashcards
(39 cards)
AP2
Clathrin adaptor
4x adaptin subunits
PICK1 binds alpha appendage
Clathrin binds beta appendage
Competitively competes with NSF for binding to GluA2
GST Pulldown
To see whether 2 proteins can interact (in a test-tube)
Can use to identify binding site
Fusion protein plasmid - GST + protein 1
Plasmid taken up by bacteria - expresses the fusion protein
Bacteria are good at making GST proteins, therefore good at making fusion proteins
Bind GST fusion protein to glutathione-coated agarose beads
Incubate with protein of interest (2)
Centrifuge
SDS-Page + Western blot - AB protein of interest (2)
2nd AB w/ chemiluminescence enzyme
Expose to photographic film to detect light - dark band = protein of interest
Ensure ABs are validated
Good for testing optimal conditions for a reaction to occur
ie. Buffer - with/without ATP, b-SNAP etc.
Co-immunoprecipitation
To see if 2 proteins interact in native tissue
Protein of interest bound to AB on sepharose bead covered in protein A/G
Incubate in brain lysate
Centrifuge
SDS-Page + Western blotting
Immunocytochemistry co-localisation
To see if 2 proteins are in the same place within a cell
Fix with formaldehyde (in order to access IC proteins)
Primary AB for protein 1 w/ fluorescence dye
Primary AB for protein 2 w/ fluorescence dye
Confocal microscopy - overlap, different colour = co-localising
Confocal = limited to the wavelength of light ~180 nm
Transfection
- Virus ie. Sindbus, lentivirus = engineer to encode virus; encourage viral DNA to incorporate into host cell’s DNA
- Lipofection = liposome associates with DNA encoding protein of interest; forms a DNA-lipid complex; lipid fuses with PM, releases DNA
- Biolistics = DNA-coated gold bullets
Control - gold w/ no DNA; ensure gold is inert
Show that LTD is mediated by clathrin-mediated endocytosis
Disrupt amphiphysin-dynamin
Amphiphysin associates with the neck of the clathrin-coated pit, recruits dynamin (GTPase) via SH3 domains which pinches off the newly formed vesicle
Memory requiring LTD
Novel object recognition memory = perihinal cortex
Blocked w/ in vivo expression of AP2-GluA2 peptide
Memory requiring LTP
Spatial object memory = hippocampus
Forgetting = GluA2-internalisation
-promoted natural forgetting with peptide: GluA2-NSF
-prevent long-term natural forgetting with peptide: GluA2-AP2 (no endocytosis)
Promote endocytosis
Peptide to disrupt: GluA2-NSF
NSF stabilises AMPARs at the synapse
In vivo memory tasks
Disrupt GluA2-AP2
- memory disrupted in the perihinal cortex (novel recognition memory)
- speed up natural forgetting process in the hippocampus (spatial memory -object location memory)
Disrupt GluA2-NSF (stabilise AMPARs at the synapse)
-prevent natural long-term memory loss
Forgetting in the hippocampus occurs due to GluA2-containing AMPARs being internalised!
The persistence of learning depends on the maintenance of steady-state level of synaptic GluA2-containing AMPARs = requires interaction with NSF-GluA2
Disrupt GluA2-NSF
GluA2-NSF interaction discovered via yeast 2-hybrid scanning
Promote endocytosis (NSF cannot stabilise AMPARs at the synapse)
Occlude LTD - therefore involved in mechanism of LTD!
Disrupt GluA2-AP5
Inhibit GluA2-endocytosis
Endosomal markers
Early endosome = EEA1
Recycling endosomes = Rab11, Syntaxin13
Late endosomes = LAMP1
Which interactions compete for the same binding site?
GluA2-NSF (stabilise AMPARs at synapse)
GluA2-AP2 (involved in clathrin-mediated endocytosis)
What amino acids do kinases require to recognise a phosphorylation site?
Example!
Lysine residue
ie. LTD = phosphorylation @ Ser880 on GluA2
Block P from occuring my mutating lysine resiue (K–>A) = reduced LTD
Need phosphorylation at Ser880 for FULL LTD to occur!
Phosphorylation of Ser880
P @ Ser880 on GluA2 occurs during LTD
By PKC:
For = phorbol esters increase phosphospecific AB staining
Against = PKC inhibition (ie. CalC/BIS) does not stop P-Ser880 or LTD from occuring
Could be - multiple kinases act to P-Ser880!
Inhibit phosphorylation = reduce LTD
Capacitance studies = GluA2 is recycled faster to the synapse (PICK1 cannot bind GluA2 + retain from the synapse)
Hypothesis: PICK1 binds PKC
-interaction brings PKC close to GluA2 to P-Ser880 - PICK1 acts as a scaffold for PKC
BUT - PICK1 K/O - P-Ser880 still occurs!
Future - try inhibiting PICK1-PKC binding site (avoid problems associated with knocking-out proteins)
PICK1-GluA2 = Ca relationship
Peak ~ 15uM
Biphasic - could explain how increasing [Ca] results in either LTD or LTP
Smaller [Ca] increase = LTD
Larger [Ca] increase = LTP
***timings are also important;
LTD = prolonged, modest increase
LTP = transient, large increase
It is possible that a Ca-sensing protein will respond to specific patterns of Ca accumulation over time
Deletion of NTD (glutamate + aspartate aa) = no biphasic relationship - no LTD occurring - need Ca sensor to trigger LTD
Need PICK1 to bind Ca (+ dimerise) in order to drive internalisation of AMPARs - therefore no LTD can occur
Ca binding PICK1
Binds to region near NTD
Acidic amino acids = glutamate + aspartate
Circular dichromism studies (with/without Ca bound) + thermal denaturation studies = decrease in intramolecular bonds + destabilisation of tertiary interactions
Expose BAR domains = can dimerise
DIFFERENT TO EXO = proteins involved contain C2 domains!
BAR domains
Highly conserved dimerisation domains - involved in membrane dynamics
Contains +vely charged lysine residues - mediate binding to the -vely charged phospholipid heads
ie. PICK1, amphiphysin
GluA2-NSF
Interaction stabilises GluA2-containing AMPARs at the surface
NSF binds to a juxtamembrane region of GluA2
Co-factor = alpha-SNAP (binds to PICK1’s BAR domain)
SNAPs act as a physical connection with NSF and PICK1!
The mechanical force of ATP hydrolysis can be transferred, via SNAPs, to dissociate PICK1-GluA2 = ‘rotational shearing’ effect
It is unclear whether PICK1 dimers dissociate into monomers = maybe try FRET experiments???
GluA2-NSF interaction
- Enhanced via nitrolyslation!!!
- Ca-sensitive: basal Ca favours binding; high [Ca] blocks interaction (LTD) - allows PICK1-mediated endo to occur
~15uM
-optimal GluA2-PICK1 binding
-inhibits GluA2-NSF
(remember NSF competes with AP2 binding to GluA2)
Inhibiting GluA2-NSF = occludes LTD (see a rundown in EPSC prior to LFS)
LTD
P @ Ser880 on GluA2 by PKC
Inhibition of GluA2-NSF
Experimentally inducing LTP
High frequency stimulation/theta burst stimulation
Glycine
Experimentally inducing LTD
Low frequency stimulation
NDMA (activate extra-synaptic receptors)
Co-factor to NSF
Alpha-SNAP
Deletion of alpha-SNAP = embryonic death
NSF+SNAP regulate PICK1-GluA2 interactions AND they regulate the SNARE complex
Beta-SNAP = Block effect - stops NSF from working
